DESCRIPTION (taken from the application): The objective of this project is to explain the molecular mechanisms by which the myosin light chain kinase (MLCK) phosphorylates the regulatory light chain of myosin (LC20) and thus regulates the smooth muscle contraction. The experiments are designed to test the hypothesis that MLCK forms extensible, flexible crossbridges between the thin and thick filaments, which phosphorylate the myosin heads sequentially on the thick filaments during filament sliding. Four types of experiments will be performed: 1) The affinity and stoichiometry of MLCK binding to F-actin will be measured in the presence and absence of tropomyosin, caldesmon and myosin. The full-length MLCK and the N-terminal fragments of various lengths will be used. Site specific and zero-length crosslinking will be used to identify the site(s) of interaction on F-actin. 2) The flexibility of synthetic analogs of the N-terminal repetitive segment of the mammalian MLCK in solution will be studied using resonance energy transfer technique. 3) The kinetics of phosphorylation of LC20 alone and in myosin thick filaments by intact MLCK and by MLCK truncated at the N-terminus (no actin binding site) or the C-terminus (no myosin binding site) or both will be measured in the absence and presence of actin filaments. Antibody specific for the phosphorlated LC20 will be used to map the distribution of phosphorylation myosin heads in smooth muscle strip upon their activation at fixed lengths and upon shortening. 4) The effects of phosphorylation by protein kinases ERK2 and PAK on the actin, myosin and calmodulin binding properties of MLCK and on its enzymatic activity will be evaluated. The results of our studies will provide explanation for some important features of the thick filament-linked regulation in smooth muscle. They will also shed light on the complex relation between the Ca2+ -dependent and Ca2a+ -independent regulation of contraction in smooth muscle.